Modulation of HBsAg expression on the cell surface of human hepatoma cells in culture continuously exposed to monoclonal anti HBs

259MODULATION OF HBsAg EXPRESSIONON THE CELL SURFACEOF HUMANHEPATOMACELLS IN CULTURE CONTINUOUSLY EXPOSEDTO MONOCLONALANTI HBs. D. Shouval and J.R. Wands*, The Liver Research Laboratory, Department of Medicine A, Hadassal University Hospital, Jerusalem, Israel and The Gastrointestinal Unit*, Mass. General Hospita Boston, Mass. USA. We have previously shown that administration of mouse monoclonal antibodies to HBsAg effectively protects about 70% of nude mice against PLC/PRF/5 hepatoma tumor formation. However, 5-10% of tumor injected mice do not respond favorably to immunotherapy and develop giant tumors that escape monoclonal anti HBs therapy. The present study was designed to reconstruct this "breakthrough phenomenon" in v i t r o in order to investigate the mechanism involved. PLC/PRF/5 human hepatoma cells t-]Tat secrete HBsAg were grown in culture in the continuous presence of monoclonal anti HBs of the IgG2a and IgM isotypes at concentrations of 125 and 25 ~g/lO5 cells/ml respectively. Cells were treated once a week with rabbit complement, then trypsinized and reseeded. Treated and untreated control PLC/PRF/5 cells were propagated for 15 passages in culture and surviving cells were cloned. Monoclonal anti HBs treated cells .(breakthrough clones) were then tested for t h e i r capacity to bind 125I labeled monoclonal anti HBs antibodies. Binding of labeled IgG2a anti HBs to the IgG2a treated breakthrough clone was reduced 4 and 7 folds as compared to untreated or IgM treated hepatoma cell clones respectively. A similar pattern, although less pronounced was observed for binding of labeled monoclonal IgM anti HBs to IgM treated breakthrough clones. We failed to grow PLC/PRF/5 cells treated simultaneously with IgG2a, IgM anti HBs and complement for more than 5-7 passages, since a l l cells were destroyed. These data indicate that continuous treatment of human hepatoma cells persistently infected by hepatitis B virus with monoclonal antibodies to HBsAg of d i s t i n c t s p e c i f i c i t i e s may modulate HBsAg expression on the cell surface and cause the breakthrough phenomenon. Results support the hypothesis that the breakthrough phenomenon observed in vivo is preventable provided different monoclonal antibodies are used sequentially or s i m u ~ a ~ s ly for immunotherapy.

260 A N E P I D E M I C P.

Skinh~j,

OF

CIRRHOSIS

J.

Petersen

M e d i c a l D e p a r t m e n t A, Copenhagen, Denmark.

IN D A N I S H

& T.

WOMEN

Thorsen

Rigshospitalet

& Council

on

Alcohol

and

Narcotics,

A s t u d y w a s p e r f o r m e d o n a n o u t b r e a k of l i v e r d a m a g e in m i d d l e a g e d w o m e n w i t h r e g a r d to p o s s i b l e c a u s e s a n d l o n g t e r m s e q u e l e . Medico-statistical data from death certificates, notified viral hepatitis and on a l c o h o l c o n s u m p t i o n were re-evaluated. T h e o u t b r e a k a p p e a r e d as an e x p l o s i v e g r o w t h in f a t a l i t i e s f r o m s u b a c u t e liv e r f a i l u r e or c i r r h o s i s in D a n i s h w o m e n in t h e m i d 1 9 4 0 ' s a m o u n t i n g to a tot a l of 1 5 0 0 ( 7 5 / 1 0 0 . 0 0 0 ) . M o r t a l i t y d u e to c i r r h o s i s remained subsequently h i g h c o m p a r e d to m e n d u e to a c o n t i n u e d excess mortality in t h e c o h o r d o f wom e n w h o in 1 9 4 5 w e r e o v e r t h e a g e of 45 y e a r s . U p to 1981 t h i s g r o u p s h o w e d a n e x c e s s of 4 . 0 0 0 f a t a l i t i e s . The available data show that the disease cann o t be e x p l a i n e d b y the e p i d e m i c h e p a t i t i s (A?) o c c u r i n g in 1 9 4 2 - 4 4 o r by increased consumption o f a l c o h o l . P o s s i b l e c a u s e s c o u l d be a p h a r m a c e u t i c a l p r o d u c t or a d d i t i v e . A l t e r n a t i v e l y an unique Non A non B viral hepatitis was transiently introduced into the Danish population.

$130